Sub-functionalization and epigenetic regulation of a biosynthetic gene cluster in Solanaceae

Biosynthetic gene clusters (BGCs) are sets of often heterologous genes that are genetically and functionally linked. Among eukaryotes, BGCs are most common in plants and fungi, where they ensure the co-expression of the different enzymes coordinating the biosynthesis of specialised metabolites. Here, we report on a newly identified withanolide BGC in Physalis grisea (ground-cherry), a member of the nightshade family (Solanaceae). A combination of transcriptomic, epigenomic and metabolic analyses, revealed that, following a duplication event, this BGC evolved two tissue-specifically expressed sub-clusters, containing eight pairs of paralogs that contribute to related but distinct biochemical processes; this sub- functionalisation is tightly associated with epigenetic features and the local chromatin environment. The two sub-clusters appear strictly isolated from each other at the chromatin structural level, each forming a highly self-interacting chromatin domain with tissue-dependent levels of condensation. This correlates with gene expression exclusively in either above- or below-ground tissue, thus spatially separating the production of different withanolide compounds. By comparative phylogenomic analysis, we show that the withanolide BGC most likely evolved before the diversification of the Solanaceae family and underwent lineage- specific diversifications and losses. The tissue-specific sub-functionalisation is common to species of the Physalideae tribe but distinct from other, independent duplication events outside of this clade. In sum, our study reports on the first instance of an epigenetically modulated sub-functionalisation within a BGC and sheds light on the biosynthesis of withanolides, a highly diverse group of steroidal triterpenoids important in plant defence and amenable to pharmaceutical applications due to their anti-inflammatory, antibiotic and anti- cancer properties.